This invention relates to an adsorbent medium and to its use in retaining large molecular species, e.g. in the purification of DNA.
DEAE (diethylaminoethane) has been used to derivatise Sepharase or Sephadex beads to provide an anion exchange resin, having quaternary ammonium groups. This is available from Pharmacia as DEAE-streamline. It is used as an anticholesteremic.
Cellulosic sponge materials have been used in various forms as the basis of an adsorbent medium, typically after the introduction of ion-exchange groups. For example, GB-A-1226448 discloses the introduction of cross-linking residues into regenerated cellulose, together with or followed by the introduction of ion-exchange groups. The resultant medium can be used, packed in a column. Various physical forms are disclosed.
WO-A-9117830 discloses an adsorbent medium (HVFM) prepared by cross-linking a flexible hydrophilic sponge that contains fibrous, hydrophilic reinforcement, and introducing functional groups. The cross-linking is controlled so that the resulting sponge has a water retention value of 2 to 6. It is proposed that this medium is suitable for the isolation or separation of macromolecules such as proteins, while retaining mechanical strength. However, in addition to its low water retention value, this medium has poor flow properties.
A particular area in which an efficient, commercial process is required, for purifying samples, is in the preparation of pharmaceutical grade plasmid DNA for gene therapy. Although many processes have been developed for the large-scale production of recombinant plasmid DNA, e.g. using E. coli as the host cell, purification of the product is necessary before the functional gene can be used, i.e. for expression in somatic tissues with the intention of selectively correcting or modulating disease conditions.
Purification of plasmid DNA has traditionally been on a very small scale for research purposes. Purification of plasmid DNA for preclinical toxicology, human clinical trials, and ultimately for an approved pharmaceutical indication, requires a process that reproducibly meets all the quality and regulatory standards and can be used to purify large quantities of the material economically.
The standard method for plasmid DNA purification by molecular biologists has been by caesium chloride/ethidium bromide ultracentrifugation. This method is unacceptable for the production of clinical materials, because it uses mutagenic reagents and is unscalable.
Alternative methods for the purification of plasmid DNA have been developed using a combination of chromatography techniques. Problems associated with such techniques have been low capacity of the adsorbents for plasmid DNA, denaturation and breakdown of the DNA molecule, and losses due to filtration of the feedstock containing plasmid DNA. Problems associated with filtration have been resolved by using the adsorbents in fluidised bed mode, but this requires specially designed hardware for large-scale applications.
This invention is based on the discovery that an adsorbent medium comprising particles of a cellulose sponge material carrying functional groups, can be effectively used to retain species having molecular weight of at least one million Daltons, e.g. viruses and DNA. In particular, it has been found that an effective adsorbent can be prepared from readily-available sponge material, after removal of fibrous reinforcement.
The new porous matrix has high capacity for plasmid DNA, and can be used in a packed bed with unfiltered feedstock. It can be used in any type of chromatographic technique, including ion-exchange, and also affinity and hydrophobic interaction.